PROJECT SUMMARY The long-term goal of this project is to understand how epithelial stem cells in the skin establish distinct patterns of gene expression during their differentiation into specialized cell lineages and how these genetic programs are altered in pathological skin conditions associated with impaired cell differentiation. Research into genome and chromatin biology has revealed that in addition to signaling/transcription factor- dependent regulatory mechanisms, lineage-specific gene expression programs are also regulated epigenetically, i.e., via regulation of covalent DNA/histone modifications and higher-order chromatin remodeling. DNA methylation and subsequent oxidation of 5-methylcytosine into 5-hydroxymethylcytosine (5hmC) are key epigenetic events regulating development and stem cell differentiation in mammals. Oxidation of 5- methylcytosine is catalyzed by the TET1/2/3 family enzymes and serve as an important step in DNA demethylation. Recent data reveal that Tet proteins plays essential roles in many biological processes including development, cancer and cellular reprogramming, while genetic ablation of all three Tet genes is lethal. However, the role of Tet-mediated DNA hydroxymethylation in the control of gene expression in keratinocytes during terminal differentiation in the epidermis and hair follicle remain obscured. Our preliminary data reveal that 5hmC-modified DNA is abundant in the hair follicle bulge, as well as in differentiating epidermal and hair matrix keratinocytes. Furthermore, genetic Tet3 ablation results in alterations of epidermal barrier formation during embryonic development, while Shh-Cre mediated ablation of all three Tet genes in the hair matrix keratinocytes results in alterations of the hair shaft structure and hair keratin gene expression. In this proposal, we will test the hypothesis that Tet proteins serve as critical determinants that regulate gene expression programmes in differentiating epidermal and hair follicle keratinocytes via DNA demethylation at the promoters and enhancers of lineage-specific genes, as well as of key genes that control epidermal barrier formation and hair follicle cycling. This hypothesis will be addressed via two Specific Aims: 1) Define the roles of Tet1, Tet2 and Tet3 in the control of epidermal development, terminal keratinocyte differentiation and epidermal barrier maintenance. 2) Define the common features and differential impact of the distinct Tet genes on self-renewing and differentiation potentials of the hair follicle epithelial stem cells and their progenies during physiological hair cycle-dependent skin regeneration. This project will have a fundamental impact on our current knowledge of epigenetic mechanisms that regulate genome reorganization in stem cells during their differentiation and will promote the progress towards the development of novel paradigms for treatment of skin disorders via targeting Tet enzymes and epigenome.